Cryoprotectant Treatment Research Articles

Assessing the viability of cryopreserved coconut zygotic embryos by electrolytic conductivity and potassium leaching

The objective of this work was to adapt the application of electrolytic conductivity and potassium leaching tests to assess the viability of cryopreserved embryos of 'Anão Verde do Brasil de Jiqui' (AVeJBr) coconut. The zygotic embryos were excised, sterilized and subjected to four cryoprotectant treatments combined with three incubation times (12, 16 and 20 hours), totaling 12 treatments. The pre-treatment of mature zygotic embryos of AVeJBr coconut using a cryoprotectant with 1.75 mol L-1 of sucrose + 15% glycerol for 12 and 16 hours promoted lower embryo humidity and increased viability in electrolytic conductivity and potassium leaching tests. Samples with ten embryos are sufficient for electrolytic conductivity analysis in cryopreserved or non-cryopreserved AVeJBr coconut zygotic embryos. The 4 to 8 hour imbibition period of the embryos is promising for the electrolytic conductivity analysis of non-cryopreserved mature zygotic embryos of AVeJBr coconut.

Pig oocyte vitrification by cryotop method: Effects on viability, spindle and chromosome configuration and in vitro fertilization

Three experiments were designed to evaluate the effects of vitrification using Cryotop method on MII porcine oocyte viability, chromosomes configuration, meiotic spindle morphology and in vitro fertilization; to do this, in vitro matured oocytes were subjected to the cryoprotectant treatment excluding the plunging into liquid nitrogen, the whole vitrification/warming/rehydration procedure or no treatment (control). In experiment 1 viable oocytes were not reduced by either cryoprotectants or vitrification when they were evaluated immediately after warming and cryoprotectant dilution. However, after a 2 h incubation, the survival rate significantly decreased ( P < 0.05). In experiment 2 cryoprotectant exposure significantly ( P < 0.05) influenced spindle morphology even if chromosome organization did not vary, while vitrification significantly ( P < 0.05) increased oocytes with damaged spindles and chromosomes displaced from the metaphase plate. No significant improvements in these parameters were observed after 2 h of incubation but, on the contrary, the rate of oocytes with normal chromosome configuration was reduced. In experiment 3 significant differences among the three groups in the fertilization rate but not in the percentages of monospermy fertilization were recorded; in addition, exposure to cryoprotectants and vitrification significantly ( P < 0.05) increased degenerated oocyte rate. Overall, these findings confirm that porcine oocytes at MII stage are very sensitive to vitrification, which reduces the rate of viable oocytes and alters microtubule organization, thus impairing fertilization; in addition, incubation of oocytes for 2 h after devitrification seems to be detrimental rather than ameliorative. Further improvements of the current protocol will be necessary in order to optimize the Cryotop method for vitrifying pig matured oocytes.

1I1636 完全酸化型ウシ心筋チトクロム酸化酵素の吸収スペクトルへの結晶化、及び凍結防止処理の影響(ヘム蛋白質 1,第49回日本生物物理学会年会)

1I1636 完全酸化型ウシ心筋チトクロム酸化酵素の吸収スペクトルへの結晶化、及び凍結防止処理の影響(ヘム蛋白質 1,第49回日本生物物理学会年会)

A novel method of cryopreservation without a cryoprotectant for immature somatic embryos of conifer

Cryopreservation of embryogenic tissue is an essential storage step in genotype selection and seedling production through somatic embryogenesis. To date, immature conifer somatic embryos, at the proliferation step, were only able to tolerate ultra low temperature after prior cryoprotectant treatments. We report a novel cryopreservation method for conifer (interior spruce and Douglas-fir) embryogenic tissue focusing on the maturation step of developing embryos that forgoes such cryoprotectant treatment. In this study, somatic embryos matured on culture media containing abscisic acid (ABA) at 20°C for 8 weeks. Typically, matured embryos in this manner were able to survive cryopreservation. The embryogenicity, however, decreased with increasing embryo maturity. Non-freezing low temperatures, such as 5°C, not only inhibited cotyledon development but also maintained embryogenicity. Cryotolerance was successfully induced when embryos were matured (or pretreated) under 5°C for a suitable culture period, typically 4–8 weeks. These embryos were able to survive a rapid cooling process and liquid nitrogen storage without the addition of any cryoprotectants. After cryopreservation, embryogenic tissue was recovered in both interior spruce and Douglas-fir. Embryo maturation tests indicated no difference in mature embryo yields with or without cryopreservation in interior spruce. The key factors inducing cryotolerance included ABA supplementation in culture media and low temperature pretreatment. Optimum combinations of these factors can result in high rates of tissue survival and high embryogenicity after cryopreservation.

135. Proteomic analysis of in vitrovanilla ( Vanilla planifolia) apices subjected to cryoprotective treatments following vitrification approach

135. Proteomic analysis of in vitrovanilla ( Vanilla planifolia) apices subjected to cryoprotective treatments following vitrification approach

Fertile plant regeneration from cryopreserved calli of Oryza rufipogon Griff. and assessment of variation in the progeny of regenerated plants

A protocol was developed for preservation of calli of Oryza rufipogon Griff. in liquid nitrogen (-196°C). Optimal conditions for cryoprotection, pre-freezing and post-thaw recovery of calli were identified. Cryoprotectant treatment at low temperature, pre-freezing in isopropanol bath for 1h at -70°C, rapid thawing and proper removal of cryoprotectant were critical for post-thaw survival. Genetic fidelity of the R1 plants obtained from cryopreserved, cryoprotectant-treated and untreated calli was assessed by phenotypic and molecular characterization. Comparison of phenotypic characters with seed-derived control plants showed no significant variation in the agronomic characters, but seed physical characters showed significant reduction in all the in vitro generated plants. Molecular data generated using 26 rice simple sequence repeat markers showed 4.78-7.25% change from control. Results suggested that both callus induction and cryopreservation induced heritable variations in O. rufipogon. In addition, a combination of phenotypic and molecular characterization using an appropriate marker provided better insight into genetic fidelity of recovered plants.

Comparative analysis of transcriptional responses to the cryoprotectants, dimethyl sulfoxide and trehalose, which confer tolerance to freeze–thaw stress in Saccharomyces cerevisiae

We have used microarray analysis to monitor the gene expression profile of Saccharomyces cerevisiae BY4743 in the presence of the cryoprotectants, dimethyl sulfoxide (Me 2SO) and trehalose. Analysis of these profiles suggests that both cryoprotectants increased the expression of genes involved in protein synthesis, ribosomal biogenesis, fatty acid biosynthesis, ergosterol biosynthesis, cell wall biosynthesis, and cellular accumulation of low molecular compounds such as glycerol, arginine and proline. Cryoprotectant treatment reduced the expression of genes involved in the β-oxidation of fatty acids. In addition, Me 2SO increased the expression of genes involved in protein refolding and trehalose increased the expression of genes involved in spore formation. This study supported that exposure to cryoprotectants prior to freezing not only reduce the freeze–thaw damage but also provide various process to the recovery from freeze–thaw damage.

Effects of Cryoprotectants and Post-storage Priming on Seed Germination of Sugi (Cryptomeria japonica

Abstract Among the ex situ methods for the preservation of forest tree germplasm, conventional seed banking is considered the most efficient method for the majority of species whilst cryopreservation has an important role in long-term conservation. The influence of cryoprotectants prior to liquid nitrogen (LN) storage and osmoconditioning priming treatments with polyethylene glycol (PEG) after LN storage were evaluated for germination (%), germination value (GV) and mean germination time (MGT) in seeds of Cryptomeria japonica. Sugi seeds were treated with two cryoprotectants (DMSO and PVS2) before immersion into LN and stored for three days. Although the C. japonica seeds survived liquid nitrogen treatment, their viability was reduced from 25% of intact seeds to 17% in LN. The seeds treated with 35% DMSO showed higher germination (28%) as compared with seeds directly-immersed in LN without cryoprotectant treatment. In contrast, PVS2 pretreated seeds decreased germination (13%). Two concentrations of PEG priming treatments were carried out. Osmoconditioning treatment in -0.4 MPa PEG solution after removal from LN enhanced the subsequent germination percentage and decreased MGT. PEG treatments at both -0.4 and -1.2MPa to PVS2 pretreated seeds before cryopreservation partially alleviated the negative effect of PVS2. DMSO pretreated seeds immersed into LN and treated with -0.4MPa PEG maintained the 28% of germination obtained in the control conditions and reduced the mean germination time (MGT). It can therefore be summarized that pretreatment with 35% DMSO was suitable for the cryopreservation of C. japonica seeds whilst the PEG priming treatment circumvented imbibitional damage after LN exposure.

160. Effect of cryoprotectant treatment and chilling on oxidative stress in zebrafish (Danio rerio) early ovarian follicles

160. Effect of cryoprotectant treatment and chilling on oxidative stress in zebrafish (Danio rerio) early ovarian follicles

152. Optimization of protein extraction method for proteomic analysis of vanilla apices subjected to cryoprotective treatments

152. Optimization of protein extraction method for proteomic analysis of vanilla apices subjected to cryoprotective treatments

Production of good-quality porcine blastocysts by in vitro fertilization of follicular oocytes vitrified at the germinal vesicle stage

We investigated survival, meiotic competence, cytoplasmic maturation, in vitro fertilization, and development of immature porcine (Sus scrofa) oocytes cryopreserved by a modified solid surface vitrification protocol. Cumulus-oocyte complexes (COCs) collected from follicles 3 to 6 mm in diameter in abattoir-derived ovaries of prepubertal gilts were either vitrified (Vitrified group), subjected to cryoprotectant treatment (CPA group), or used without any treatment (Control group). Oocyte viability was assayed by staining with fluorescein diacetate. Live oocytes were matured in vitro and their meiotic progression investigated by nuclear staining. In a series of experiments, the glutathione (GSH) content of in vitro–matured oocytes and viability of cumulus cells were assayed simultaneously. The in vitro–matured oocytes were also fertilized and cultured in vitro to assess their ability to be fertilized and to develop to the blastocyst stage, respectively. The proportion of viable oocytes in the Vitrified group was significantly lower than that in the CPA and Control groups (27.7%, 90.4%, and 100%, respectively). Among the three groups, there were no differences in meiotic competence, cumulus viability, and GSH levels at the end of in vitro maturation. Fertilization parameters (i.e., rates of male pronucleus formation, monospermy, and second polar body extrusion) were also similar among groups. However, comparison of the developmental abilities of oocytes in the Vitrified, CPA, and Control groups revealed that the Vitrified group had a significantly reduced ability to undergo first cleavage (34.4%, 63.3%, and 69.0%) and to develop to the blastocyst stage (5.1%, 25.5%, and 34.6%). The mean total cell numbers in blastocysts after 6 d of culture were not significantly different among the Vitrified, CPA, and Control groups (40.3, 42.8, and 43.4). In conclusion, despite low survival rates and impaired development in the Vitrified group, meiotic competence, cytoplasmic maturation, and subsequent fertilization characteristics of surviving germinal vesicle oocytes were unaffected by vitrification, and high-quality blastocysts were produced from vitrified immature oocytes.

Evaluation of glycerol removal techniques, cryoprotectants, and insemination methods for cryopreserving rooster sperm with implications of regeneration of breed or line or both

A series of experiments was designed to evaluate the quality of cryopreserved rooster sperm and its fertility so that programs needing to bank germplasm and recreate animals can do so utilizing a minimal amount of cryopreserved semen. In experiment 1, rooster semen from the National Animal Germplasm Program genebank was thawed and glycerol was removed using a discontinuous Accudenz column or by stepwise dilution. The postthaw sperm motilities, plasma membrane integrity, and concentration were determined before and after deglycerolization. Line differences in postthaw sperm concentration and progressive motility were observed before deglycerolization (P < 0.05). After glycerol removal, the sperm that was centrifuged through Accudenz had greater total motility (37 vs. 33% sperm; P < 0.05), but use of the stepwise dilution method recovered more sperm per milliliter (320.4 × 106) compared with the Accudenz method (239.2 × 106 sperm; P < 0.05; range across 6 lines of 165.7 to 581.0 × 106 sperm/mL). In experiment 2, rooster semen was cryopreserved using Lake’s diluent containing either dimethyl acetamide (DMA) or glycerol as the cryoprotectants. Postthaw analysis revealed that the samples cryopreserved with glycerol survived freezing better, determined by total motility (47.8 and 15.1% glycerol and DMA samples, respectively; P < 0.05) and annexin V analyses (1.6 and 11.3% membrane-damaged sperm for glycerol and DMA samples, respectively; P < 0.05). Differences in sperm motilities (total and progressive motility) and velocities (path velocity, straight-line velocity, curvilinear velocity) were observed between the 2 cryoprotectant treatments once the glycerol had been removed from those samples cryopreserved with glycerol, of which the glycerol samples had significantly more motile sperm and higher velocities (P < 0.05). The fertility of the samples frozen using the 2 cryoprotectants was tested using a single insemination (intravaginal or intramagnal) of 200 × 106 sperm and the fertility (number of live embryos) was evaluated over 18 d. Overall, the intravaginal inseminations had lower fertility than the intramagnal inseminations (P < 0.05). In the intravaginal inseminations, the sperm cryopreserved using DMA resulted in lower fertility, but there were no differences in fertility in the intramagnal inseminations due to cryoprotectant (P > 0.05). These results indicate that reasonable postthaw sperm quality and fertility can be derived using cryopreserved rooster semen. By utilizing this information, estimations can be made for storing sufficient material for line or breed, or both, recreation programs.

Soy lecithin replaces egg yolk for cryopreservation of human sperm without adversely affecting postthaw motility, morphology, sperm DNA integrity, or sperm binding to hyaluronate

Semen specimens (one ejaculate from each of 20 consenting study participants) were subjected to routine semen analysis, an in vitro sperm binding assay (HBA), and a sperm chromatin dispersion assay (HaloSperm), both before and after cryopreservation using cryoprotectant media supplemented with either egg yolk or soy lecithin. Comparing the equivalency of the two phospholipid cryopreservation supplements with regard to postthaw functional parameters demonstrated that there were no statistically significant differences between the two supplements for [1] recovery of motile sperm, [2] maintenance of sperm cell morphology, [3] maintenance of the ability of sperm to bind to hyaluronate in vitro, or [4] maintenance of sperm DNA integrity.

Pollination of Dendrobium Hybrids Using Cryopreserved Pollen

Pollination effectiveness was evaluated for pollen (pollinia) from two Dendrobium hybrids, ‘Sena Red’ and ‘Mini WRL’, submitted to cryopreservation using a vitrification protocol. Parameters evaluated included pollinia exposure to a previtrification solution (PVS2) under ice (0 °C) or room (27 ± 2 °C) temperatures from 1 to 4 hours before cryopreservation (LN). On removal from cryopreservation, pollinia were used to pollinate flowers of the same hybrids to verify viability and germination. All pollinia showed high percentages of germination (greater than 80%) after crosses were performed, except for pollinia from Dendrobium ‘Sena Red’ submitted to 3 hours of precooling (0 °C) in PVS2 followed by LN (60%) and for pollinia submitted to PVS2 for 3 hours at room temperature with no precooling (70%). Capsules were formed for both hybrids and seeds were successfully produced. The seed viability test revealed high viability (90% to 95%) for all treatments for both hybrids. Seeds observed under a microscope contained well-formed embryos and no abnormalities were identified. Seeds from all treatments germinated. Germinating seeds developed into healthy seedlings with well-formed leaves and roots. Cryopreservation of pollinia was successfully accomplished either by direct storage in liquid nitrogen without cryoprotection treatments or by using a PVS2 vitrification protocol.

Cathepsin activities and membrane integrity of zebrafish ( Danio rerio) oocytes after freezing to −196 °C using controlled slow cooling

This study investigated enzymatic activity of cathepsins and the membrane integrity of zebrafish ( Danio rerio) oocytes after freezing to −196 °C using controlled slow cooling. Stage III oocytes (>0.5 mm), obtained through dissection of anaesthetised female fish and desegregation of ovarian cumulus, were exposed to 2M methanol or 2 M DMSO (both prepared in Hank’s medium) for 30 min at 22 °C before being loaded into 0.5 ml plastic straws and placed into a programmable cooler. After controlled slow freezing, samples were plunged into liquid nitrogen (LN) and held for at least 10 min, and thawed by immersing straws into a 27 °C water bath for 10 s. Thawed oocytes were washed twice in Hank’s medium. Cathepsin activity and membrane integrity of oocytes were assessed both after cryoprotectant treatment at 22 °C and after freezing in LN. Cathepsin B and L colorimetric analyses were performed using substrates Z-Arg-ArgNNap and Z-Phe-Arg-4 MβNA-HCl, respectively, and 2-naphthylamine and 4-methoxy-2-naphthylamine were used as standards. Cathepsin D activity was performed by analysing the level of hydrolytic action on haemoglobin. Oocytes membrane integrity was assessed using 0.2% Trypan blue staining for 5 min. Analysis of cathepsin activities showed that whilst the activity of cathepsin B and D was not affected by 2 M DMSO treatment, their activity was lowered when treated with 2M methanol. Following freezing to −196 °C, the activity of all cathepsins (B, D and L) was significantly decreased in both 2 M DMSO and 2 M methanol. Trypan blue staining showed that 63.0 ± 11.3% and 72.7 ± 5.2% oocytes membrane stayed intact after DMSO and methanol treatment for 30 min at 22 °C, respectively, whilst 14.9 ± 2.6% and 1.4 ± 0.8% stayed intact after freezing in DMSO and methanol to −196 °C. The results indicate that cryoprotectant treatment and freezing modified the activities of lysosomal enzymes involved in oocyte maturation and yolk mobilisation.

Plasmolysis and recovery of different cell types in cryoprotected shoot tips of Mentha × piperita

Successful cryopreservation of plant shoot tips is dependent upon effective desiccation through osmotic or physical processes. Microscopy techniques were used to determine the extent of cellular damage and plasmolysis that occurs in peppermint (Mentha x piperita) shoot tips during the process of cryopreservation, using the cryoprotectant plant vitrification solution 2 (PVS2) (30% glycerol, 15% dimethyl sulfoxide, 15% ethylene glycol, 0.4 M sucrose) prior to liquid-nitrogen exposure. The meristem cells were the smallest and least plasmolyzed cell type of the shoot tips, while the large, older leaf and lower cortex cells were the most damaged. When treated with cryoprotectant solutions, meristem cells exhibited concave plasmolysis, suggesting that this cell type has a highly viscous protoplasm, and protoplasts have many cell wall attachment sites. Shoot tip cells were most severely plasmolyzed after PVS2 treatment, liquid-nitrogen exposure, and warming in 1.2 M sucrose. Successful recovery may be dependent upon surviving the plasmolytic conditions induced by warming and diluting treated shoot tips in 1.2 M sucrose solutions. In peppermint shoot tips, clumps of young meristem or young leaf cells survive the cryopreservation process and regenerate plants containing many shoots. Cryoprotective treatments that favor survival of small, meristematic cells and young leaf cells are most likely to produce high survival rates after liquid-nitrogen exposure.

Influence of Semen Storage and Cryoprotectant on Post-thaw Viability and Fertility of Stallion Spermatozoa

The aim of the current study was to verify that stallion spermatozoa could be cooled for 24 hours and then frozen. In experiment I, one ejaculate from each of 13 stallions was used. Semen was collected and split into two parts; one part immediately frozen using standard cryopreservation techniques and the other diluted, stored in an Equitainer for 24 hours, and then frozen. In experiment II, one ejaculate from each of 12 stallions was collected, diluted with Botu-Semen, and split into two parts: one cooled in an Equitainer and the other in Max-Semen Express without prior centrifugation. After 24 hours of cooling, the samples were centrifuged to remove seminal plasma and concentrate the sperm, and resuspended in Botu-Crio ® extender containing one of three cryoprotectant treatments (1% glycerol + 4% dimethylformamide, 1% glycerol + 4% dimethylacetamide and 1% glycerol + 4% methylformamide), maintained at 5°C for 20 minutes, then frozen in nitrogen vapor. No difference was observed between the two cooling systems. The association of 1% glycerol and 4% methylformamide provided the best post-thaw progressive motility. For experiment III, two stallions were used for a fertility trial. Forty-three inseminations were performed using 22 mares. No differences were seen in semen parameters and pregnancy rates when comparing the two freezing protocols (conventional and cooled/frozen). Pregnancy rates for conventional and cooled/frozen semen were, respectively, 72.7% and 82.3% (stallion A), and 40.0% and 50.0% (stallion B). We concluded that cooling equine semen for 24 hours before freezing, while maintaining sperm viability and fertility, is possible.

A Method for Cryopreserving Chicken Primordial Germ Cells

This study established a method for preserving chicken primordial germ cells (PGC) that enables long-term storage in liquid N. Gonads were harvested from stage 27 chick embryos and pooled in groups of 5, 10 (10E), or 20 embryos, contributing gonads to the cell suspension. The gonadal cells, including PGC, were then frozen in 1 of the following cryoprotectant treatments: 2.5% dimethyl sulfoxide (DMSO), 5% DMSO, 10% DMSO, 2.5% ethylene glycol (EG), 5% EG, 10% EG, and 0% cryoprotectant as a control. The cells were liberated and frozen in a biosecure cryopreservation straw at a rate of -1 degrees C/min until reaching -85 degrees C and were then plunged into liquid N (-196 degrees C), in which they were stored until analysis. Flow cytometry was used to analyze the PGC post-thaw. The PGC marker stage-specific embryonic antigen-1, which was detected with goat antimouse IgM fluorescein isothiocyanate, was used to label all PGC, and propidium iodide was used to detect cells with compromised cell membranes. There was an interaction effect for the number of viable PGC per individual embryo (P < or = 0.05). The highest level (183.6 +/- 28.4) of viable PGC per individual embryo was observed for 10% EG with 10E and was significantly higher (P < or = 0.05) than cryopreservation in 2.5% DMSO with 10E and 20 embryos, 2.5% EG with 10E, 5% EG with 10E, and all 0% cryoprotectant treatments. No statistical interaction (P > 0.05) was observed for the percentage of viable PGC. However, the highest percentage (80.6%) was observed at 10% EG with 10E. It was demonstrated that PGC were successfully frozen, and the most effective treatment was 10% EG with 10 embryos/straw.

Assessment of gamete quality for the eastern oyster ( Crassostrea virginica) by use of fluorescent dyes

Evaluation of sperm motility is the single most widely used parameter to determine semen quality in mammals and aquatic species. While a good indicator for fresh sperm viability, post-thaw motility is not always effective at predicting fertilizing ability. Techniques using fluorescent dyes can assess functionality of mammalian sperm, but have not been widely applied in aquatic organisms. The eastern oyster Crassostrea virginica is an important mollusk in the United States, and cryopreservation protocols have been developed to preserve sperm and larvae to assist research and hatchery production. In this study, protocols were developed to assess sperm cell membrane integrity and mitochondrial function by flow cytometry and to assess viability of eggs by fluorescence microscopy. The fluorescent dyes SYBR 14 and propidium iodide (PI) (to assess membrane integrity) and rhodamine 123 ( R123) (to assess mitochondrial membrane potential) were used to evaluate the quality of thawed oyster sperm previously cryopreserved with different cryoprotectant and thawing treatments. Membrane integrity results were correlated with motility of thawed sperm and mitochondrial membrane potential with fertilizing ability. Fluorescein diacetate (FDA) was used to assess cytotoxicity of cryoprotectant solutions and post-thaw damage to oyster eggs. The results indicated that membrane integrity ( P = 0.004) and thawing treatments ( P = 0.04), and mitochondrial membrane potential ( P = 0.0015) were correlated with motility. Fertilizing ability was correlated with cryoprotectant treatments ( P = 0.0258) and with mitochondrial membrane potential ( P = 0.001). The dye FDA was useful in indicating structural integrity of fresh and thawed eggs. Exposure of eggs, without freezing, to dimethyl sulfoxide yielded higher percentages of stained eggs and fertilization rate than did exposure to propylene glycol ( P = 0.002). Thawed eggs were not stained with FDA (<1%) and larvae were not produced.

Development of a new rapid measurement technique for fish embryo membrane permeability studies using impedance spectroscopy

Information on fish embryo membrane permeability is vital in their cryopreservation. Whilst conventional volumetric measurement based assessment methods have been widely used in fish embryo membrane permeability studies, they are lengthy and reduce the capacity for multi-embryo measurement during an experimental run. A new rapid ‘real-time’ measurement technique is required to determine membrane permeability during cryoprotectant treatment. In this study, zebrafish (Danio rerio) embryo membrane permeability to cryoprotectants was investigated using impedance spectroscopy. An embryo holding cell, capable of holding up to 10 zebrafish embryos was built incorporating the original system electrods for measuring the impedance spectra. The holding cell was tested with deionised water and a series of KCl solutions with known conductance values to confirm the performance of the modified system. Untreated intact embryos were then tested to optimise the loading capacity and sensitivity of the system. To study the impedance changes of zebrafish embryos during cryoprotectant exposure, three, six or nine embryos at 50% epiboly stage were loaded into the holding cell in egg water, which was then removed and replaced by 0.5, 1.0, 2.0 or 3M methanol or dimethyl sulfoxide (DMSO). The impedance changes of the loaded embryos in different cryoprotectant solutions were monitored over 30min at 22°C, immediately following embryo exposure to cryoprotectants, at the frequency range of 10–106Hz. The impedance changes of the embryos in egg water were used as controls. Results from this study showed that the optimum embryo loading level was six embryos per cell for each experimental run. The optimum frequency was identified at 103.14 or 1380Hz which provided good sensitivity and reproducibility. Significant impedance changes were detected after embryos were exposed to different concentrations of cryoprotectants. The results agreed well with those obtained from conventional volumetric based studies.